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Live-Cell Imaging Increases Impact on R&D

Live-cell imaging technologies are providing what is often the first detailed look at intracellular interactions within living cells, yielding fresh insights into molecular functions and dynamic structures that may change within fractions of a second or during days of repeated interrogation. Speakers at CHI’s “Live Cell Imaging” meeting to be held later this month in Boston will discuss how FRET, FLIM, OLID, and other technologies are bringing live cells to light.

Gerard Marriott, Ph.D., professor of bioengineering at the University of California, Berkeley, is developing a fluorescence imaging technique that enhances contrast, thereby revealing new detail about living cells. Called optical lock-in detection (OLID) imaging microscopy, this technique generates a modulated fluorescence signal from a new class of optical switch that can be isolated from background signals.

“A typical cell harbors about 10,000 equivalents of the green fluorescence protein that acts as a background signal,” Dr. Marriott says. Traditionally, that background noise is eliminated by chemically treating the sample, but that kills live cells, he explains. Dr. Marriott’s approach involves rapidly and reversibly inter-converting the two distinct (A and B) states of an optical switch. “Only the B state is capable of fluorescing,” he points out.

He has developed synthetic optical switches and used genetically encoded proteins that behave as optical switches. “We use a short pulse of near-ultraviolet light to convert the A state to the B state, and then irradiate the sample with visible light to convert the B state back to the A state,” he explains.

One cycle is A-B-A. Doing this repeatedly, for 5 to 20 cycles, creates a modulated fluorescence intensity profile from the B state that is unique to that optical switch. The modulated signal of an optical switch-labeled protein can be digitally filtered for every pixel in the image from the unmodulated background fluorescence. Dr. Marriott says this method yields dramatic improvements in image contrast, revealing detail that otherwise is hidden in the background.

Most of the work so far has involved proof of concept in aqueous solutions and living cells and tissues. Now, Dr. Marriott’s lab is generating a second-generation OLID microscope and improved optical switches for use in animals and, ultimately, humans.

“The goal is to control the two states of an optical switch using two-photon light at 800 nm wavelength or longer, that will penetrate several hundred microns under skin,” he says, which increases scanning speed. “Completing one cycle in 10 microseconds is feasible and would allow us to complete an optical switching study within 50 microseconds.” That’s fast enough to see the dynamics of single protein complexes within living cells.

Dynamic Interactions

At Indiana University School of Medicine, Richard N. Day, Ph.D., professor, department of cellular and integrative physiology, uses FRET-based microscopy to measure the network interactions of transcription factors and coregulatory proteins within the nuclear compartment of cells. Unlike biochemical assays that disrupt cellular structures, FRET provides evidence for protein interactions in the intact living cells.

To use this technique, Dr. Day explains, researchers “label the proteins of interest with either donor or acceptor fluorescent proteins, and look for the transfer of excitation from the donor to the acceptor protein.” This method, however, also detects background fluorescence, which makes the occurrence of FRET difficult to prove. Options include performing acceptor photo-bleaching, which dequenches the donor signal, and fluorescent lifetime imaging, which measures the time the protein remains excited.

“For the first time, we have a way of looking at how a particular disease might affect specific protein interactions inside living cells,” Dr. Day says. “We’re asking how specific protein domains mediate interactions between proteins, and how genetic mutations disrupt these interactions.”

Dr. Day’s lab is measuring the homeodomain transcription factor Pit-1, which is involved in developmental processes, and the CCAAT/enhancer binding protein alpha (C/EBPα) in the nucleus of living mouse pituitary cells. The group is also monitoring dynamic interactions between C/EBPα and the heterochromatin protein-1 alpha (HP1α) in regions of the centromeric heterochromatin in pituitary cells. “That study allows us, at the molecular level, to see the effect of changes in particular amino acid sequences,” Dr. Day says. The next phase of his research will address multiple protein-to-protein interactions involved in chromatin remodeling and gene regulation.

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